JP2018501483A - Method for reducing interference - Google Patents

Abstract

The present invention is a method for determining an analyte in a sample suspected of containing an analyte, comprising: a) contacting the sample with at least first and second capture compounds for the analyte, The first and second capture compounds are non-identical capture compounds, and the capture compounds compete in binding to the analyte; b) the capture compound contacted with the sample is identified with a specific agent The specific agent competes with the analyte for binding to the capture compound; c) determines the amount of complex comprising the specific agent and capture compound; and d) It relates to said method comprising the step of determining said analyte in the sample based on the result of step c). The present invention further includes a method for improving the specificity of an indirect immunoassay for determining an analyte, comprising replacing at least 10% of a capture compound with a non-identical capture compound; The capture compound relates to the method, wherein the capture compound competes with the introduced capture compound for binding to the analyte. The invention further relates to kits, devices and uses related to the methods described above.

Description

  The present invention is a method for determining an analyte in a sample suspected of containing an analyte, comprising: a) contacting the sample with at least first and second capture compounds for the analyte, The first and second capture compounds are non-identical capture compounds, and the capture compounds compete in binding to the analyte; b) the capture compound contacted with the sample is identified with a specific agent The specific agent competes with the analyte for binding to the capture compound; c) determines the amount of complex comprising the specific agent and capture compound; and d) It relates to said method comprising the step of determining said analyte in the sample based on the result of step c). The present invention further includes a method for improving the specificity of an indirect immunoassay for determining an analyte, comprising replacing at least 10% of a capture compound with a non-identical capture compound; The capture compound relates to the method, wherein the capture compound competes with the introduced capture compound for binding to the analyte. The invention further relates to kits, devices and uses related to the methods described above.

Background Art Laboratory tests, especially immunological tests, have become valuable tools in the diagnosis of diseases. Immunoassays have particularly offered the possibility of specifically detecting a single analyte or group of analytes in a complex mixture, such as a body fluid, such as blood or plasma. However, some causes of interference in immunoassays, such as cross-reactivity of capture compounds with interfering substances, non-specific binding of detector compounds to the solid phase, capture compounds and detection compounds to name a few “Bridged” binding has been identified by heterophilic antibodies or especially human anti-mouse antibodies (HAMA) (eg, Park & Kricka (2013), Chapter 5.3—Interferences in Immunoassay, The Immunoassay Handbook (4th)). Edition), supervised by David Wild, Elsevier, Oxford: 403; Schitetecate (2012), Interferences in Immunoassays, Advances in Immu Noassay Technology, Dr. Norman HL Chiu (supervised)).

  In competitive immunoassays, the analyte contained in the sample competes with the labeled analyte (specific agent) for binding to the capture compound, which is often an antibody or in a serum sample, for example, When trying to test for the presence of antibodies against a pathogen, it is the antigen of said pathogen. If the analyte concentration in the sample is high, there is strong competition leading to a decrease in binding of the specific agent to the capture compound, causing a signal decrease, which leads to a quantitative or qualitative test result, depending on the test format . It is known in the art that an antigen used as a capture compound can also be bound by a sample-derived interfering compound, which is a disadvantage of immunoassays. These interfering compounds compete with specific agents for the binding epitope of the capture compound. Depending on how severe the competition is, false test results can occur, causing a decrease in the specificity of each immunoassay. This reduction in specificity can be particularly noticeable when using complex capture compounds such as viral capsids, even consisting of several subunits that potentially contain a large number of conformational epitopes. In such cases, classical methods of interference cancellation, such as the addition of alternative targets for interfering compounds, can be inefficient.

  In a non-competitive immunoassay, the analyte is contacted with a compound that specifically binds to the analyte and either itself carries the label or is the target of a second molecule that carries the label. To detect the analyte. Thus, in a noncompetitive immunoassay, the amount of analyte is determined by determining the amount of complex formed between the analyte and the detection compound carrying the label. Thus, as described above, similar specificity problems may be encountered.

  Antigens that are only slightly different immunologically (eg production in different expression systems, different amino acid sequences, differences in glycosylation, differences in purification and refolding methods, and differences in buffering and storage conditions) Have a range. Thus, a sample that leads to a false result in an immunoassay with antigen X1 may provide the correct result in a test with antigen X2, and vice versa. Thus, exchanging antigens for different antigens often does not lead to improved specificity and only changes the range of samples that lead to false results.

  Accordingly, it is an object of the present invention to provide an improved immunoassay avoiding the problems described above.

  These problems are solved by the methods, kits, devices and compositions having the features of the independent claims. Exemplary embodiments that can be achieved in a single manner or in any arbitrary combination are listed in the dependent claims.

Accordingly, the present invention is a method for determining an analyte in a sample suspected of containing the analyte, comprising:
a) contacting the sample with at least a first and second capture compound for the analyte, wherein the first and second capture compounds are non-identical capture compounds, and the capture compound is the analyte Competing in binding to;
b) contacting the capture compound contacted with the sample with a specific agent, wherein the specific agent competes with the analyte for binding to the capture compound;
c) determining the amount of complex comprising the specific agent and capture compound; and d) determining the analyte in the sample based on the result of step c).

  The terms “having”, “including” or “included” as used below, or any arbitrary grammatical variations thereof, are used in a non-exclusive manner. Thus, these terms include, in addition to the features introduced by these terms, both situations where there are no additional features and situations where one or more additional features are present in the entities described in this background. It is also possible to point. By way of example, the expressions “A has B”, “A contains B” and “A contains B” are situations where A has no other elements in addition to B (ie alone And the situation consisting exclusively of B), and entity A has in addition to B one or more additional elements, such as element C, elements C and D, or even a situation where there are even more elements. It is also possible to point.

  Further, the terms “preferably”, “more preferably”, “most preferably”, “specifically”, “more specifically”, “specifically”, “more specifically” as used below. ”Or similar terms are used in combination with optional features without limiting another possibility. Accordingly, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. The present invention can also be implemented using other features, as those skilled in the art will recognize. Similarly, features introduced by "in an embodiment of the invention" or similar expressions are not subject to any limitation with respect to another embodiment of the invention, without any limitation on the scope of the invention, and other features of the invention. It is intended that the feature be an optional feature without any restrictions on the possibility of combining features introduced as in the case or non-case feature. The term “about” means in the context of a particular value or ratio of the present invention said value or ratio +/− 30%, +/− 20%, +/− 10%, or in one embodiment a predetermined value or ratio. Of +/− 5%.

  In one embodiment, the method of the present invention is an in vitro method. Furthermore, steps may be included in addition to those specifically mentioned. In particular, step b) can include providing a sample, or step c) can include the addition of additional compounds to facilitate binding and detection. In addition, some or all of the processes can be assisted by automated equipment.

  As used herein, the term “determining” refers to determining in a sample at least one immunological characteristic of an analyte to be determined by the method of the present invention. An immunological feature according to the present invention is in one aspect a structural feature of an analyte that facilitates detection of the analyte in a sample by immunological means. In one embodiment, the immunological feature facilitates identification of the analyte by immunological means, and in a further embodiment facilitates quantification. Thus, typical immunological features are those that facilitate distinguishing the analyte from other chemical compounds in the sample. In one embodiment, the determination of the analyte establishes whether the analyte is present or absent in the sample at a concentration that exceeds the detection limit of the method. Methods for determining the detection limit are known to those skilled in the art. In a further aspect, the determination is semi-quantitative or quantitative determination of the amount or concentration of the analyte in the sample. For quantitative determination, the absolute or exact amount of analyte is determined, or the relative amount of analyte is determined. The relative amount can be determined when the exact amount of the analyte cannot or cannot be determined. In such cases, it may be determined whether the amount of analyte present is increasing or decreasing with respect to a second sample containing a second amount of the analyte.

  As will be appreciated by those skilled in the art, typically the first capture compound / analyte and the second capture compound / analyte complex will not need to be determined separately. Thus, in one embodiment, the first capture compound and analyte complex, and the second capture compound and analyte complex together, ie between the first and second capture compounds. Determine without distinction. Thus, in one embodiment, the total amount of analyte present in the complex comprising either the first capture compound or the second capture compound is determined.

  As will be further understood by those skilled in the art, the determination of the analyte / capture compound complex will depend on the assay format chosen. In one embodiment, the assay is a competitive immunoassay, typically a competitive heterogeneous immunoassay, i.e., the analyte competes with a labeled derivative of the analyte for binding to a capture compound bound to a solid surface, and An immunoassay that determines the amount of labeled derivative of the analyte bound to the capture compound. In one embodiment, the competition assay comprises anti-HBc, anti-HAV (anti-hepatitis A virus), anti-HBe (anti-hepatitis B e antigen), folic acid, folate RBC (red blood cells), anti-TSH-R, total vitamin D, Assays for vitamin B12, thyroxine T4, FT4 (free thyroxine), thyroxine T3, FT3 (free triiodothyronine), testosterone, progesterone, digitoxin, anti-TG, anti-TPO (antithyroid peroxidase), DGEA, or estradiol. In a further embodiment, the immunoassay is a detection compound as specified herein below, wherein a bivalent analyte, eg, an antibody, binds to a capture compound bound to a solid surface and to the analyte / capture compound complex. Is a double antigen sandwich assay (“DAGS”) that determines the amount of the analyte / capture compound complex by binding. In one embodiment, the DAGS assay comprises anti-toxoplasma IgG, anti-rubella IgG, anti-HBs 1G, HCV (hepatitis C), eg HCVII, CMV (cytomegalovirus) IgG, syphilis, HTLV (human T cell lymphoproliferative virus) ), Or an assay for Chagas (American trypanosomiasis).

  The term “biological molecule” is known to those skilled in the art and typically relates to a molecule produced by metabolism of at least one organism. Thus, the term “biological macromolecule” relates in one aspect to a polymer produced by an organism from a monomeric precursor. Typical biological macromolecules are polypeptides, DNA, RNA or polysaccharides.

The term “analyte” as used herein binds to a capture compound and / or detection compound of the invention with sufficient affinity to allow detection of the analyte / capture compound and / or detection compound complex. Chemical molecule, in one aspect, relates to an organic molecule. In one embodiment, the dissociation constant (K _d ) of the analyte / ligand complex has a maximum of 10 ⁻⁷ mol / L, in a further embodiment, a maximum of 10 ⁻⁸ mol / L, and in a further embodiment, a maximum of 10 ⁻⁹ mol / L. L. In one embodiment, the dissociation constant of the complex formed between the first capture compound of the present invention and the analyte, and the dissociation of the complex formed between the second capture compound of the present invention and the analyte. The constants do not differ by more than 5 times; in one embodiment, do not differ by more than 2 times; in further embodiments, do not differ by more than 1.5 times. In one embodiment, the dissociation constant of the complex formed between the first capture compound and the analyte is between 70% and 130 of the dissociation constant of the complex formed between the second capture compound and the analyte. % Value. In a further embodiment, the dissociation constant of the complex formed between the first capture compound and the analyte is between 80% and 120% of the dissociation constant of the complex formed between the second capture compound and the analyte. Has the value of In a further embodiment, the dissociation constant of the complex formed between the first capture compound and the analyte is between 90% and 110% of the dissociation constant of the complex formed between the second capture compound and the analyte. Has the value of In further embodiments, the dissociation constant of the complex formed between the first capture compound and the analyte and the dissociation constant of the complex formed between the second capture compound and the analyte are essentially equal. Or equal. In one embodiment, the analyte has a molecular weight of at least 100 (corresponding to 100 atomic mass units, and 100 Da; 1 Da corresponds to 1.66 × 10 ⁻²⁷ kg), in a further embodiment, at least 250, a further embodiment. Having a molecular weight of at least 500, or in a further embodiment, at least 1000. In one embodiment, the analyte is a biological molecule, and in a further embodiment, the analyte is a biological macromolecule. In a further embodiment, the analyte is a polypeptide.

  In one embodiment, when the analyte of the invention is a polypeptide, the polypeptide is against an antigen produced by an infectious pathogen, such as a virus or bacteria, or an antibody, such as an antigen produced by an infectious pathogen. An antibody produced by a subject. In one embodiment, the analyte is a polypeptide, in a further embodiment an antibody against a viral antigen, in a further embodiment an antibody against a viral polypeptide, or in a further embodiment an antibody against a viral capsid polypeptide. In one embodiment, the viral capsid polypeptide is a hepatitis virus capsid polypeptide, in one embodiment hepatitis B virus (HB) capsid polypeptide, or in a further embodiment, an HB core (HBc) antigen. Thus, in one embodiment, the analyte is an antibody against a hepatitis virus capsid polypeptide, eg, against a hepatitis B virus (HB) capsid polypeptide, typically against an HB core (HBc) antigen or an HBe antigen. In one embodiment, the first capture compound is an HBc antigen as shown in Genbank accession number Q17UT2 GI: 1238677942 (SEQ ID NO: 1). In a further embodiment, the first capture compound is an HBc antigen as shown in Genbank accession number Q17UT2 GI: 1238677942 (Hepatitis B virus subtype adw2 core antigen, SEQ ID NO: 1), and the second capture compound is Genbank accession number P03147.1 GI: 116947 (hepatitis B virus genotype D subtype adw core antigen, SEQ ID NO: 2). In further embodiments, the first and second capture compounds are HBcAg identified by Genbank accession numbers P03148.3 GI: 1662115076, P03149.1 GI: 116945, NP_6477607.1 GI: 21326588, and AY1232424.1 GI: 22203511. An HBc antigen sequence selected from HBc antigen sequences known in the art, such as a protein sequence.

  In one embodiment, to facilitate cloning, the N and / or C terminus of each HBV core antigen sequence may be shortened by deleting 1 to 5 N and / or C terminal amino acids of the antigen sequence. Good. In a further embodiment, a tag comprising 2-15 amino acids can be added to either the N-terminus or C-terminus of the antigen or to both ends without interfering with the antigenic properties of the HBV core antigen.

The term polypeptide herein includes, in one embodiment, variants and fragments of the polypeptide specifically indicated. Variants specifically include, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99 of the amino acid sequence set forth in SEQ ID NO: 1 or Polypeptides comprising amino acid sequences that are% identical are included. The percent identity value is preferably calculated over the entire amino acid sequence region. A series of programs based on various algorithms are available to those skilled in the art for comparing different sequences. In this context, the Needleman and Wunsch or Smith and Waterman algorithms yield particularly reliable results. To perform sequence alignment, the program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989: 151-153) or the programs Gap and BestFit [Needleman and Wunsch (J. Mol. Biol. 48; 443-453 (1970)) and Smith and Waterman (Adv. Appl. Math. 2; 482-489 (1981))], which are part of the GCG software packet [Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 (1991)]. The sequence identity values referred to above, expressed in percent (%), shall preferably be determined using the program GAP over the entire sequence region with the following settings: gap load: 50, long Thickness load: 3, average match 10.000 and average mismatch: 0.000, unless otherwise specified, shall always be used as a standard setting for sequence alignment. Polypeptides comprising are also included in one embodiment as the polypeptides of the present invention. The fragment may also be a polypeptide having that of a capture compound or detection compound as specified above. Thus, a polypeptide can comprise or consist of a domain of a polypeptide of the invention that confers the biological activity. A fragment as meant herein preferably comprises at least 20, at least 30, at least 50, at least 80, at least 100 or at least 150 contiguous amino acids of any one of the foregoing amino acid sequences. Any at least 50, at least 100, at least 250 or at least 500 consecutive amino acid residues.

  The polypeptides of the present invention either consist essentially of the aforementioned amino acid sequence or comprise the aforementioned amino acid sequence. Thus, they may also contain additional polypeptide sequences. In particular, the polypeptide of the invention may be a fusion protein, wherein one partner of the fusion protein is a polypeptide as listed above. Such fusion proteins can be used as additional moieties, such as fatty acids or other enzymes in the lipid biosynthetic pathway, polypeptides for monitoring expression (eg, green, yellow, blue or red fluorescent proteins, alkaline phosphatase, etc.) or detectable markers. Or may include a so-called “tag” that may serve as a supplementary reference for purification purposes or to attach the polypeptide to a solid surface. Tags for different purposes are well known in the art and include FLAG tags, 6-histidine tags, MYC tags, biotin and the like.

However, the analyte is a low molecular weight compound that can be determined by complex formation with a capture compound including, for example, thyroxine (T4), triiodothyronine (T3), folic acid, folic acid binding protein, vitamin B ₁₂ , intrinsic factor, etc. It is also assumed that there is.

  As used herein, the term “capture compound” binds directly or indirectly to an analyte of the invention, as specified herein above, and is bound to or bound to a solid surface. It relates to chemical molecules that are adapted to do.

  In one embodiment, the capture compound is an organic molecule, in a further embodiment a biological macromolecule as specified herein above, for example a polypeptide as specified above. In one embodiment, the capture compound binds indirectly to the analyte of the invention with sufficient affinity to allow detection of a complex comprising the analyte and the capture compound; that is, in such cases, the capture compound Is an indirect ligand. The term “indirectly binds” as used herein is a chemical molecule in which the ligand does not directly contact the analyte, but is bound specifically to the analyte in one embodiment. With respect to binding in contact with a chemical molecule, in one embodiment, the molecule that is bound to the analyte is a molecule that is directly bound to the analyte, ie, a direct ligand. Thus, in one embodiment, the analyte, the chemical molecule bound to the analyte, and the indirect ligand form a complex with the properties as indicated above, particularly with the dissociation constant as indicated. . As will be appreciated by those skilled in the art, the definition of the term “competing in binding” applies to the indirect ligands of the present invention, mutatis mutandis, ie, an indirect ligand is an embodiment. In the method, it is impossible to simultaneously bind to the molecule bound to the analyte. In further embodiments, the capture compound binds directly to the analyte of the invention with sufficient affinity to allow detection of the analyte / capture compound complex, as specified herein above. Thus, in one embodiment, the capture compound is a direct ligand.

  In accordance with the present invention, at least first and second capture compounds are provided, wherein the first and second capture compounds are non-identical capture compounds. As used herein, the term “non-identical” capture compound relates to two or more species of capture compound molecules that have measurable differences in at least one chemical and / or physical property. Typically, the at least one chemical and / or physical property is not an indicator of, and not a property of, the binding of the capture compound to a solid surface. Typical properties are amino acid sequence, glycosylation, three-dimensional folding and / or conformation, and polypeptide chain length. However, differences in impurity concentration and / or identity in two preparations of the same capture compound are also contemplated by the present invention. Thus, in one embodiment, the second capture compound comprises (i) introducing at least one amino acid exchange within the amino acid sequence of the first capture compound, and (ii) the first capture compound. Producing the second capture compound in a different cell background when compared to (iii) removing glycosylation of the second capture compound when compared to the first capture compound. (Iv) purifying the second capture compound by different means when compared to the first capture compound; (v) comparing to the first capture compound. Under different conditions, the second capture compound may be denatured and / or refolded, and (vi) different when compared to the first capture compound. Under conditions, by at least one to store the second capture compound of may or obtained obtained from the first capture compound. In one embodiment, the first and second capture compounds are antibodies that recognize essentially the same epitope, and the second capture compound is a variant of an antibody that is the first capture compound. is there. In a further embodiment, at least one of the first and second capture compounds is not an antibody. In another embodiment, the first and second capture compounds are not antibodies.

  In accordance with the present invention, at least first and second capture compounds are provided, wherein the capture compounds compete for binding to the analyte. As used herein, the term that indicates that two compounds “compete in binding” to an analyte means that the molecules of the compound do not bind simultaneously to essentially the same binding site of the analyte. Regarding the characteristics that are possible. Thus, typically, when two capture compounds compete for binding to an analyte, and if the analyte has one binding site for the capture compound, one molecule of the capture compound at each time point Only can bind to the analyte. One skilled in the art will appreciate that the above is true where it is to be changed and has two or more binding sites for the capture compound. Those skilled in the art know how to determine competition in binding. In one embodiment, the competition for binding to the analyte is binding of the analyte to the same or essentially the same substructure. In a further embodiment, when the analyte is a polypeptide, the epitope to which the first capture compound binds and the epitope to which the second capture compound binds are at least three amino acids common to the analyte, in one embodiment 3 Has two consecutive amino acids. Typically, in such cases, the epitope to which the first capture compound binds and the epitope to which the second capture compound binds have at least 4, 5, 6, or 7 amino acids common to the analyte.

  In one embodiment, the first and second capture compounds do not compete for binding to the interfering compound. The term “interfering compound” as used herein relates to a compound that reduces the specificity of the immunoassay. In one embodiment, the interfering compound is a non-analyte compound that binds to the capture compound of the invention.

  In one embodiment, the first and second capture compounds are used in a ratio of 1: 5 to 5: 1, typically 1: 2 to 2: 1, about 1: 1, or 1: 1. In further embodiments, three capture compounds are used in a ratio of about 2: 1: 1, 1: 2: 1, or 1: 1: 2, about 1: 1: 1, or 1: 1: 1. Thus, in one embodiment, the first and second capture compounds and any additional capture compounds that are potentially present are present in the reaction mixture in approximately the same amount, and in further embodiments, in the same amount.

  Methods for attaching biological molecules, typically polypeptides, to solid surfaces are well known in the art and include, for example, hydrophobic interactions, binding by biotinylation, and immobilized streptavidin, covalent bonds, antibodies Including binding through antigen interactions, etc., or combinations of these interactions, such as antibody-antigen interactions between antibodies and pathogen polypeptides, wherein the antibodies are biotinylated and are immobilized on a solid surface through immobilized streptavidin Are connected. Accordingly, the capture compound is preferably also a capture complex. In one embodiment, the capture compound is a compound of a capture complex that binds directly to the analyte. Those skilled in the art know how to bind capture compounds or complexes to a solid surface, depending on the solid surface selected. In one embodiment, the capture compound is a viral polypeptide, such as a viral capsid polypeptide. In one embodiment, the capture compound is a hepatitis virus capsid polypeptide, and in one embodiment, a hepatitis B virus (HB) capsid polypeptide, such as an HB core (HBc) antigen or an HBe antigen. However, it is also envisioned that the capture compound is an antibody.

  The term “antibody” as used herein refers to a monoclonal antibody, a multispecific antibody (eg, a bispecific antibody) formed from at least two intact antibodies, and another herein. Antibody fragments are included so long as they exhibit the desired binding activity as specified at the site. In one embodiment, the antibody is not an antibody contained in an antiserum, typically not a polyclonal antibody or a polyclonal serum. Thus, in one embodiment, the antibody is an antibody contained in a mixture, wherein at least 80%, in one embodiment at least 90%, and in a further embodiment at least 95% of the antibody molecules contained in said mixture At least one capture compound or at least one detection compound. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a full length antibody or antibody fragment.

  Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies (immunoglobulins) can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these are further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Is possible. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described in, for example, Abbas et al., Cellular and Mol. Immunology, 4th edition, W.M. B. Saunders, Co. (2000). An antibody may be part of a larger fusion molecule formed by the covalent or non-covalent association of an antibody with one or more other proteins or peptides.

  The terms “full length antibody”, “intact antibody” and “total antibody” are used interchangeably herein and are not substantially intact, not antibody fragments as defined below. Refers to the type of antibody. The term specifically refers to an antibody comprising a heavy chain that contains an Fc region. “Antibody fragments” comprise a portion of an intact antibody, and in one embodiment, its antigen binding region. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; Diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of the antibody includes two identical antigen-binding fragments, called “Fab” fragments, each containing a single antigen-binding site, and the remaining “Fc” fragments, reflecting their ability to easily crystallize Produce. Pepsin treatment yields an F (ab ') 2 fragment that has two antigen combining sites and is still capable of cross-linking antigen. “Fv” is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, the double chain Fv species consists of a dimer in which one heavy chain and one light chain variable domain are in tight non-covalent association. In a single chain Fv (scFv) species, one heavy chain and one light chain variable domain appear to be capable of associating in a “dimeric” structure where the light and heavy chains are similar to those in the double chain Fv species Can be covalently linked by a flexible peptide linker. In this configuration, the three hypervariable regions (HVR) of each variable domain interact to define the antigen binding site. Taken together, the six HVRs confer antigen binding specificity to the antibody. However, a single variable domain (or half of the Fv that contains only three HVRs specific for the antigen) has the ability to recognize and bind to the antigen, albeit with a lower affinity than the entire binding site. The term “diabody” refers to an antibody fragment having two antigen binding sites, the fragment comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain. (VH-VL). By using a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with the complementary domain of another chain and generates two antigen-binding sites. To do. The diabody may be bivalent or bispecific. Diabodies are described, for example, in EP 0 404 097; WO 1993/01161; Hudson et al., Nat. Med. 9 (2003) 129-134; and Hollinger et al., PNAS USA 90 (1993) 6444-6448. Triabodies and tetrabodies are also described by Hudson et al., Nat. Med. 9 (2003) 129-134.

  The term “monoclonal antibody” as used herein refers to an antibody that is obtained from a population of substantially homogeneous antibodies, ie, individual antibodies that are included in the population may be present in small amounts. It is identical except for mutations, such as naturally occurring mutations. Thus, the modifier “monoclonal” indicates that the nature of the antibody is not a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to an analyte, wherein the analyte binding polypeptide sequence is from a plurality of polypeptide sequences, Obtained by a process involving the selection of a single analyte binding polypeptide sequence. For example, the selection process may be the selection of unique clones from a pool of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. Further modification of the selected target binding sequence, eg, improving affinity for the target, humanizing the target binding sequence, improving production in cell culture, reducing in vivo immunogenicity, multispecific antibodies It is to be understood that antibodies that are capable of generating and the like, and that include a modified target binding sequence are also monoclonal antibodies of the invention. In contrast to polyclonal antibody preparations that contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of the monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibody preparation is advantageous in that it is typically free of other immunoglobulins.

  As used herein, the term “solid surface” is any suitable solid surface that is adapted for binding of the capture compounds of the invention and separated from a sample, eg, by physical means. About. In one embodiment, the solid surface is a surface of beads, in one embodiment microbeads, such as magnetic or paramagnetic microbeads. In one embodiment, the surface is adapted to improve covalent or non-covalent binding of the capture compound to a molecule that binds to the capture compound substructure, eg, by attachment. Typical molecules that bind to the substructure of the capture compound are, for example, antibodies, streptavidin, complexed nickel ions, and the like. In further embodiments, the solid surface binds to the capture compound by covalent or non-covalent bonds, for example by hydrophobic interactions. Thus, in one embodiment, the solid surface is the surface of a multi-cluster plate. In one embodiment, the surface of the multi-cluster plate is pretreated to increase the affinity and / or volume for capture compound binding. Appropriate pretreatment is known in the art.

  The term “sample” as used herein relates to a sample presumed to contain an analyte of the invention. In one embodiment, the sample is or comprises a body fluid sample, a tissue or organ-derived sample, or a wash / rinse solution or swab or smear sample obtained from an external or internal body surface. The sample, in one embodiment, includes at least one analyte as specified elsewhere herein. Samples of blood, plasma, serum, urine, saliva, or tears are included by the methods of the present invention. The sample may be obtained by brush, (cotton) swab, spatula, rinse / wash solution, punch biopsy device, use of a puncture of the cavity with a needle or lancet, or surgical instruments. However, in one embodiment, samples obtained by well-known techniques, including curettage, swab or biopsy from the genitourinary tract, perianal region, anal canal, oral cavity, airway digestive tract and epithelium are also of the present invention. Included as a sample. Cell-free fluids may be obtained from body fluids or tissues or organs by lysis techniques such as homogenization and / or by separation techniques such as filtration or centrifugation. In one embodiment, a sample is obtained from a body fluid known to contain HB virus polypeptides or / and antibodies to at least one HB virus polypeptide, ie, in one embodiment, blood, plasma, serum, saliva, and the like. It will be understood that the sample may be further processed to carry out the method of the invention. In particular, the cells may be removed from the sample by methods and means known in the art. In addition, at least one analyte may be extracted and / or purified from the sample by methods and means known in the art. Thus, the term sample can also relate to a preparation that contains or is suspected of containing at least one analyte diluted, concentrated, purified and / or extracted from the sample.

  In one embodiment, the method of the invention is a competitive immunoassay. Therefore, the method further includes the step of mixing the specific agent with the sample. In one embodiment, the method of the invention is a heterogeneous competitive immunoassay and the amount of complex formed between the analyte and two non-identical capture compounds is determined by the specific agent and the non-identical capture compounds. Determining indirectly by determining the amount of complex formed between.

  The term “contacting” is understood by those skilled in the art in the context of the method of the present invention. In one embodiment, the term relates to physically contacting a compound of the invention with a sample, or with additional compounds, thereby allowing, for example, the sample and the compound to interact.

  The term “specific agent” is known to those skilled in the art and relates to a compound that competes with an analyte for binding to a capture compound bound to an indicator. Typically, the specific agent is structurally similar to the analyte. Typically, a specific agent comprises an analyte substructure to which a capture compound bound to an indicator binds. In one embodiment, the specific agent is a compound that includes an analyte and an indicator as structural elements, or the specific agent consists of an analyte covalently bound to the indicator.

  The term “indicator” is a compound adapted herein to be able to detect the presence of a molecule or complex comprising said indicator. Typically, the indicator has detectable properties, typically optical or / and enzymatic properties. However, it is also envisioned that the detectable property is a property that releases radioactivity.

  The term “optical property” as used herein relates to any property that can be detected by an optical device. Specifically, the optically determinable property is: at least one property selected from the group consisting of: reflection property, transmission property, emission property, scattering property, fluorescence property, phosphorescence property, diffraction property, and polarization property Or include such characteristics. Further optical properties envisaged by the present invention are color, fluorescence, luminescence or refraction. In one embodiment, the optically determinable property as referred to herein is a property of an optically detectable chemical compound, such as light absorption, light emission, light reduction, or Refers to the relevant property. As used herein, detecting an optically determinable property includes detecting the presence of a previously undetectable property, detecting the absence of a previously detected property, and It will be understood to include quantitative change detection, ie, detection of a change in signal intensity that correlates to the degree of change in at least one optical property. It is understood that the term “optically detectable property” relates in one aspect to electrochemiluminescence, also known as electrogenerated chemiluminescence.

  The term “enzymatic property” as used herein relates to the property of an indicator that produces a detectable product from a substrate by a biological catalyst. Thus, enzymatic properties are typically given by the presence of a polypeptide having the enzymatic properties in the indicator. Typically, the enzymatic properties are: from the group consisting of: phosphatase activity (eg in alkaline phosphatase), peroxidase activity (eg in horseradish peroxidase), and glycosidase activity (eg in beta-galactosidase) At least one enzyme activity selected. Typical substrates for enzyme activity are well known in the art. Typically, the enzymatic activity produces a product with determinable optical properties as specified herein above, and / or the enzymatic activity produces a product that can be determined by an electrical device. To do.

  In one embodiment, the method of the invention is a double antigen sandwich assay (“DAGS”). Thus, the method comprises (i) contacting the complex with at least one detection compound, and (ii) determining the amount of a ternary complex comprising the analyte, the capture compound, and the detection compound. Further comprising detecting a complex formed between the analyte and the capture compound.

  In embodiments of the DAGS assay, a detection compound is used. The term “detection compound” as used herein binds directly or indirectly to an analyte of the invention, as specified hereinabove, and to an indicator as specified elsewhere herein. It relates to chemical molecules that are bound. In one embodiment, the detection compound is not bound to the solid surface and is not adapted to bind to the solid surface. As will be appreciated by those skilled in the art, a detection compound is an indirect detection compound, i.e., a detection compound that does not contact the analyte directly as specified hereinabove with respect to the ligand of the invention. Also good. In one embodiment, the detection compound is a direct detection compound. Thus, in one aspect, the definitions provided above for the capture compounds of the present invention apply mutatis mutandis to the detection compounds of the present invention, unless they are related to binding of the compound to a solid surface. .

  As one skilled in the art will recognize, the term “specific” means that other compounds present in the sample, typically biomolecules, are significantly different from the ligand (capture compound or detection compound) of the present invention. Used to indicate that it does not bind; as one skilled in the art will appreciate, it does not participate in the interaction with the tag that serves as a functional moiety that relaxes binding to the analyte, specific agent or solid phase It does not preclude binding of chemical compounds to regions of capture or detection compound molecules.

Furthermore, the present invention provides
(I) at least 10% of the capture compound, in one embodiment at least 25%, in further embodiments at least 40%, in further embodiments at least 45%, in further embodiments about 50% are replaced by non-identical capture compounds An indirect immunoassay for determining the analyte, wherein the displaced capture compound competes with the introduced capture compound for binding to the analyte; or (ii) at least 50 of the capture compound %, In one embodiment, at least 75%, in further embodiments, at least 90%, and in further embodiments, replacing about 100% with a non-identical capture compound, wherein the substituted capture compound is transferred to the analyte. Competing with the introduced capture compound in the binding of At least one capture compound and at least one detection compound are non-identical ligands of the analyte, or at least 50% of the detection compound, in one embodiment, at least 75%, in further embodiments, at least 90%, further embodiments In which about 100% is replaced by a non-identical detection compound, the substituted detection compound competes with the introduced detection compound for binding to the analyte, and said at least one capture compound And at least one detection compound is a non-identical ligand of the analyte,
Of a dual antigen sandwich immunoassay to determine the analyte,
It relates to a method for improving specificity.

  Typically, the percentage of capture or detection compound displaced will be selected so that a measurable effect of displacement can be theoretically expected. As will be appreciated by those skilled in the art, the expected value of a measurable effect will depend on the number of non-identical ligands used for substitution. For example, if three are used instead of one capture agent compound in the improved assay, it is preferred that, for example, 66% of the initial capture compound be replaced. As a general rule, assume that the ratio of a given capture or detection compound is (100% / n) ± 50%, where n = (number of non-identical capture or detection compounds used in the assay). Is done. In another embodiment, a ratio of (100% / n) ± 20% is used. As will be appreciated by those skilled in the art, the total percentage used will be 100%. Thus, in one embodiment, the percentage of capture or detection compound displaced is 10% to 90%, in one embodiment 25% to 75%, in further embodiments 40% to 60%, in further embodiments, About 50%.

Furthermore, the present invention provides a kit for detecting an analyte in a sample,
(I) at least two non-identical capture compounds for the analyte, wherein the capture compounds compete for binding to the analyte; or (ii) at least two non-identical detection compounds for the analyte, The kit comprising the detection compound that competes for binding to the analyte.

In a further aspect, the present invention is a kit for detecting an analyte in a sample comprising:
(I) at least two non-identical capture compounds for the analyte, the capture compounds competing for binding to the analyte, and (ii) a specific agent, wherein the analysis is for binding to the capture compound The kit includes the specific agent that competes with the product.

  The term “kit” as used herein refers to a collection of the aforementioned compounds, means or reagents of the present invention, which may or may not be packaged together. The components of the kit may be contained in separate vials (ie, as a separate part kit) or may be provided in a single vial. Furthermore, it is to be understood that the kits of the invention can be used to perform the methods referred to herein above. In one aspect, it is envisioned that all components are provided in a ready-to-use manner for performing the methods referred to above. Further, in one embodiment, the kit contains instructions for performing the method. Instructions for use may be provided by a user manual in paper or electronic form. For example, the manual can include instructions for interpreting the results obtained when performing the above-described method using the kit of the present invention. In one embodiment, a kit for detecting an analyte in a sample comprising at least two non-identical capture compounds of the present invention further comprises at least two non-identical detection compounds, wherein the detection compound comprises Competing in binding to the analyte and the capture compound does not compete with the detection compound in binding to the analyte. In further embodiments, the kit further comprises a solid support for immobilizing the capture compound or for immobilizing the analyte.

  In one embodiment, 2-10 capture compounds for the analyte, in one embodiment, 2-5 capture compounds for the analyte, in one embodiment, 2-4 capture compounds for the analyte, 1 In embodiments, a few capture compounds for the analyte are included in the kit. In further embodiments, 2-10 detection compounds for the analyte, in one embodiment, 2-5 detection compounds for the analyte, in one embodiment, 2-4 detection compounds for the analyte, one embodiment In the kit, a few detection compounds for the analyte are included in the kit.

The invention also relates to a device for determining an analyte in a sample comprising: (i) at least two non-identical capture compounds for the analyte, wherein the capture compounds compete for binding to the analyte Or (ii) at least two non-identical detection compounds for the analyte that compete for binding to the analyte, and optionally an optical indicator of the indicator contained in the detection compound; And / or said device comprising means for determining enzymatic properties.

  The term “device” as used herein relates to a system of means, including at least the aforementioned means, operably linked to each other to allow determination. Exemplary means for determining the amount of analyte and means for performing the determination are disclosed above in connection with the method of the present invention. How the means are coupled in a manner of operation will depend on the type of means included in the device. In one aspect, the means is comprised by a single device. The device thus includes (i) an analytical unit for measuring the amount of analyte in the applied sample and (ii) a computer unit for processing the data generated for the evaluation. Also good. Exemplary means for detection are disclosed in connection with aspects relating to the method of the invention above. In such cases, the means should be such that the user of the system can summarize the results of the determination of the optically and / or electrochemically determinable properties of the indicator and of the analyte by the instructions and explanations provided in the manual. The instructions are included in the device so that they are linked to be functional or, as a result of the determination, the amount or concentration of the analyte in the applied sample is output to the user. Included in possible program code. The person skilled in the art will know how to connect the means without further hassle. Typical devices are those that are applicable without the specific knowledge of a special technician, for example test strips or electronic devices that simply need to be loaded with a sample. The results may be provided as raw data output that requires interpretation by a technician. However, in one aspect, the output of the device is raw data that has been processed, ie, evaluated, and requires no technician to interpret it. Further exemplary devices are analytical units / devices such as those mentioned above (eg biosensors, arrays, solid supports coupled to ligands that specifically recognize peptides, according to the methods of the invention, Plasmon surface resonance device, NMR spectrometer, mass spectrometer, etc.) or evaluation unit / device.

  In one embodiment, 2-10 capture compounds for the analyte, in one embodiment, 2-5 capture compounds for the analyte, in one embodiment, 2-4 capture compounds for the analyte, 1 In embodiments, a few capture compounds for the analyte are included in the device. In further embodiments, 2-10 detection compounds for the analyte, in one embodiment, 2-5 detection compounds for the analyte, in one embodiment, 2-4 detection compounds for the analyte, one embodiment In which 2-3 detection compounds for the analyte are included in the device.

  Further, the present invention provides for (i) at least two non-identical capture compounds for an analyte indicative of disease for use in diagnosis, wherein the capture compounds compete for binding to the analyte; or (Ii) to a composition comprising at least two non-identical detection compounds for an analyte indicative of a disease, said detection compounds competing for binding to said analyte.

  The term “composition” as used herein refers to any composition formulated in solid, liquid or gaseous form. Said compositions comprise a compound of the invention optionally with a suitable auxiliary compound such as a diluent or carrier or further ingredients. Suitable diluents and / or carriers depend on the purpose for which the composition is to be used and other ingredients. One of ordinary skill in the art can determine such suitable diluents and / or carriers without additional hassle. In one embodiment, a composition for use in diagnosis comprising at least two non-identical capture compounds of the present invention further comprises at least two non-identical detection compounds, said detection compounds binding to said analyte And the capture compound does not compete with the detection compound for binding to the analyte.

  The term “diagnosis” as used herein refers to an assessment of the likelihood that a subject will or will suffer from a disease or condition. As will be appreciated by those skilled in the art, such assessment is usually not intended to ensure that 100% of subjects are correctly diagnosed. The term, however, requires that a statistically significant portion of a subject can be correctly diagnosed as suffering from a disease or condition. Whether or not a part is statistically significant can be determined without any hassle using various well-known statistical evaluation tools such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. Can be determined by one skilled in the art. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the possibilities envisaged by the present invention allow the diagnosis to be correct for at least 60%, at least 70%, at least 80% or at least 90% of subjects in a given cohort or population. Typical conditions to be diagnosed are for example fertility conditions or pregnancy. Typical diseases that are diagnosed are, for example, epidemic or previous infections with pathogens such as viruses, hyperthyroidism or hypofunction, vitamin deficiencies, and the like.

  Further, the present invention provides (i) first and second capture compounds that are non-identical capture compounds for determining an analyte in a sample and compete for binding to the analyte, A capture compound, or (ii) a first and second detection compound, which are non-identical detection compounds, and use of said detection compounds that compete for binding to the analyte.

Summarizing the findings of the present invention, the following embodiments are disclosed:
Aspect 1. A method for determining an analyte in a sample suspected of containing an analyte comprising: a) contacting the sample with at least first and second capture compounds for the analyte, wherein the first and second The second capture compound is a non-identical capture compound and competes for binding to the analyte;
b) the capture compound contacted with the sample,
(I) contacting with a detection compound, wherein said detection compound competes with said capture compound for binding to said analyte; or (ii) contacted with a specific agent, wherein said specific agent is said capture compound Competes with the analyte in binding to
c)
(I) determining the amount of said detection compound and capture compound, or (ii) a complex comprising said specific agent and capture compound; and d) determining said analyte in the sample based on the result of step c) Said method comprising the steps.

  Aspect 2. Competition in binding to the molecule is binding to essentially the same or the same substructure of the molecule, and in one embodiment, competition in binding to the analyte is essentially the same or the same as the analyte. Binding to a substructure, wherein in one embodiment, competition in binding to the capture compound is binding of essentially all of the capture compound in the at least one embodiment described above to the same or the same substructure. The method of embodiment 1, wherein

Aspect 3. The method of embodiment 1 or 2, wherein said non-identical capture compound is a biological molecule, in one embodiment a biological macromolecule, in a further embodiment a polypeptide.
Aspect 4. The method of any one of aspects 1-3, wherein the two non-identical capture compounds are, in one embodiment of the viral polypeptide, a variant of a viral capsid polypeptide.

  Aspect 5 The two non-identical capture compounds are variants of a hepatitis B (HB) capsid polypeptide, in one embodiment of a hepatitis virus capsid polypeptide, and in a further embodiment a variant of the HB core (HBc) antigen. Item 5. The method according to any one of Items 1 to 4.

  Aspect 6 Embodiment 1 wherein the second compound is a variant of an antibody that is a first capture compound; or the first and second capture compounds are antibodies that recognize essentially the same epitope. The method of any one of ~ 2.

Aspect 7. Said second capture compound is at least one of the following:
(I) introducing at least one amino acid exchange into the amino acid sequence of said first capture compound;
(Ii) producing the second capture compound in a different cell background when compared to the first capture compound;
(Iii) removing or preventing glycosylation of the second capture compound as compared to the first capture compound;
(Iv) purifying said second capture compound by different means and / or by different methods when compared to said first capture compound;
(V) denature and / or refold the second capture compound under different conditions when compared to the first capture compound; and (vi) the first capture compound. A method according to any one of aspects 1 to 6, wherein the method is derived from or can be derived from the first capture compound by storing the second capture compound under different conditions when compared to .

Aspect 8 The method of any one of embodiments 1-7, wherein the analyte is a biological macromolecule.
Aspect 9. The method according to any one of aspects 1-8, wherein said analyte is a polypeptide.
Aspect 10 A method according to any one of aspects 1-9, wherein said analyte is an antibody against a viral antigen, in one aspect against a viral polypeptide, in a further aspect against a viral capsid polypeptide.

  Aspect 11 Any of aspects 1-10, wherein said analyte is an antibody against hepatitis B virus (HB) capsid polypeptide, in one aspect against hepatitis virus capsid polypeptide, and in a further aspect against HB core (HBc) antigen. Or one way.

Aspect 12 The method according to any one of aspects 9 to 11, wherein the epitope bound by the capture compound has at least 3 amino acids in common.
Aspect 13 Embodiment 13. The method of any one of embodiments 1-12, wherein the determination of the analyte is a qualitative or quantitative determination of the amount of the analyte.

  Aspect 14. Step a) comprises 2 to 10 capture compounds for the analyte, in one embodiment 2 to 5 capture compounds for the analyte, in one embodiment 2 to 4 capture compounds for the analyte, in one embodiment A method according to any one of aspects 1-13, comprising contacting 2-3 of the capture compound for the analyte with the sample.

Aspect 15 Embodiment 15. The method of any one of embodiments 1-14, wherein the specific agent comprises an analyte substructure bound by a capture compound bound to an indicator.
Aspect 16. The method according to any one of aspects 1 to 15, wherein the specific agent is a compound comprising an analyte and an indicator.

Aspect 17 The method according to any one of embodiments 1 to 16, wherein the specific agent comprises an analyte covalently bound to an indicator.
Aspect 18. A method according to any one of aspects 1-17, wherein said capture compound is not contained in a polyclonal antiserum and in one aspect is not a polyclonal antibody.

Aspect 19 The method of any one of embodiments 1-18, wherein said first and second capture compounds do not compete for binding to an interfering compound.
Aspect 20
a) contacting the sample with at least a first and second capture compound for the analyte, wherein the first and second capture compounds are non-identical capture compounds, and the capture compounds to the analyte Competing in binding;
b) contacting the capture compound contacted with the sample with a specific agent, wherein the specific agent competes with the analyte for binding to the capture compound;
Any one of embodiments 1-19 comprising the step of c) determining the amount of the complex comprising the specific agent and the capture compound; and d) determining the analyte in the sample based on the result of step c). the method of.

Aspect 21. A method for determining an analyte in a sample suspected of containing an analyte comprising: a) contacting a sample with at least first and second detection compounds for the analyte, wherein the first And the second detection compound is a non-identical detection compound and the detection compound competes for binding to the analyte;
b) binding a component of the sample comprising at least the analyte to a solid surface;
c) determining the amount of complex comprising a detection compound bound to the solid surface; and d) determining the analyte in the sample based on the result of step c).

Aspect 22 Embodiment 23. The method of embodiment 22, wherein the analyte comprises an antibody and in one embodiment is an antibody.
Aspect 23. The method according to any one of aspects 21 to 22, wherein the detection compound further comprises an indicator.

  Aspect 24. The method according to any one of aspects 15-17 or 23, wherein said indicator is a compound having a detectable property, in one embodiment optical or / and enzymatic properties.

Aspect 25 25. The method of any one of embodiments 20-24, wherein said first and second detection compounds do not compete for binding to interfering compounds.
Aspect 26. A method according to any one of aspects 20 to 25, wherein step a) is performed after step b).

Aspect 27.
(A) at least 10% of the capture compounds, in one embodiment at least 25%, in further embodiments at least 40%, in further embodiments at least 45%, in further embodiments about 50% are replaced by non-identical capture compounds The substituted capture compound competes with the introduced capture compound for binding to the analyte,
(I) an indirect immunoassay for determining the analyte or (ii) a dual antigen sandwich immunoassay for determining the analyte; or (b) at least 25% of the detection compound, in one embodiment at least 40%. In a further embodiment comprising replacing at least 45%, in a further embodiment about 50% by a non-identical detection compound, wherein the substituted detection compound competes in binding of the introduced detection compound to the analyte. And a dual antigen sandwich immunoassay for determining an analyte wherein said at least one capture compound and at least one detection compound are non-identical capture compounds of said analyte,
A method to improve specificity.

  Aspect 28. Any of embodiments 1-27, wherein the chemical structure in said capture compound and / or said detection compound that binds to said analyte is the same in all said non-identical capture compounds and / or all said non-identical detection compounds One way.

Aspect 29
(I) at least two non-identical capture compounds for the analyte, in one embodiment, 2-10 capture compounds for the analyte, in one embodiment, 2-5 capture compounds for the analyte, in one embodiment 2 to 4 capture compounds for the analyte; in one embodiment, 2 to 3 capture compounds for the analyte that compete for binding to the analyte; or (ii) analysis At least two non-identical detection compounds for the analyte, in one embodiment, 2-10 detection compounds for the analyte, in one embodiment, 2-5 detection compounds for the analyte, in one embodiment, the analyte 2 to 4 detection compounds related to, in one embodiment, 2 to 3 detection compounds related to the analyte, which compete in binding to the analyte. A kit for detecting an analyte in a sample containing the detection compound.

Aspect 30 The kit of embodiment 29, further comprising a solid support for immobilizing the capture compound, or at least a component of the sample comprising the analyte.
Aspect 31
(I) at least two non-identical capture compounds for the analyte, in one embodiment, 2-10 capture compounds for the analyte, in one embodiment, 2-5 capture compounds for the analyte, in one embodiment 2 to 4 capture compounds for the analyte; in one embodiment, 2 to 3 capture compounds for the analyte that compete for binding to the analyte; or (ii) analysis At least two non-identical detection compounds for the analyte, in one embodiment, 2-10 detection compounds for the analyte, in one embodiment, 2-5 detection compounds for the analyte, in one embodiment, the analyte 2 to 4 detection compounds related to, in one embodiment, 2 to 3 detection compounds related to the analyte, which compete in binding to the analyte. A device for detecting an analyte in a sample comprising means for determining the detection compound, and optionally a means for determining the optical or / and enzymatic properties of an indicator contained in the detection compound.

Aspect 32. (I) at least two non-identical capture compounds for an analyte indicative of disease, wherein the capture compounds compete for binding to the analyte, or (ii) of the disease A composition comprising at least two non-identical detection compounds for an indicator analyte that compete for binding to the analyte.

Aspect 33. (I) at least two non-identical capture compounds for the analyte that compete for binding to the analyte; and / or (ii) the analyte for the manufacture of a diagnostic composition or diagnostic device; Use of said detection compound for at least two non-identical detection compounds with respect to binding to said analyte.

Aspect 34. The composition for use of aspect 32 or the use of aspect 33, wherein the disease is viral hepatitis.
Aspect 35. (I) a first and second capture compound that are non-identical capture compounds and compete for binding to the analyte to determine an analyte in a sample; or ( ii) use of a composition comprising said detection compound, said first and second detection compounds being non-identical detection compounds and competing in binding to said analyte.

Further optional features and aspects of the present invention will be disclosed in more detail in the following description of the aspects, in combination with the dependent claims in one aspect. Here, the features in each case can be achieved in an isolated manner, as well as in any arbitrary possible combination, as those skilled in the art will recognize. The scope of the invention is not limited by the disclosed embodiments. The embodiment is schematically shown in the figure. Here, identical reference numerals in these figures refer to identical or functionally comparable elements.
Schematic representation of the principles of the present invention as applied to a competitive assay; a: analyte, s: specific agent, c1: capture compound 1, c2: capture compound 2, i1: interfering agent 1, i2: interference Agent 2. A) The specific agent binds to capture compound 1 in the absence of analyte. B) In the presence of the analyte, the analyte binds to capture compound 1 and prevents the specific agent from binding to capture compound 1. C) In the presence of interfering agent 1 that has an affinity for capture compound 1 (but no affinity for capture compound 2), interfering agent 1 binds to capture compound 1 and thus the specific agent binds. To prevent. D) In the presence of interfering agent 2 that has affinity for capture compound 2 (but not capture compound 1), interfering agent 2 does not bind to capturing compound 1 and therefore the specific agent is capture compound 1 Is allowed to join. Conversely, E) Interfering agent 1 that has an affinity for capture compound 1 (but no affinity for capture compound 2) does not bind to capture compound 2, so that the specific agent binds to capture compound 2. And F) Interfering agent 2 binds to capture compound 2 and thus prevents specific agents from binding. When G) and H) capture compounds 1 and 2 are used in a 1: 1 ratio, for example, the false signal reduction induced by interfering agent 1 or interfering agent 2 will be reduced to approximately 50%. Schematic representation of the principles of the present invention as applied to a competitive assay; a: analyte, s: specific agent, c1: capture compound 1, c2: capture compound 2, i1: interfering agent 1, i2: interference Agent 2. A) The specific agent binds to capture compound 1 in the absence of analyte. B) In the presence of the analyte, the analyte binds to capture compound 1 and prevents the specific agent from binding to capture compound 1. C) In the presence of interfering agent 1 that has an affinity for capture compound 1 (but no affinity for capture compound 2), interfering agent 1 binds to capture compound 1 and thus the specific agent binds. To prevent. D) In the presence of interfering agent 2 that has affinity for capture compound 2 (but not capture compound 1), interfering agent 2 does not bind to capturing compound 1 and therefore the specific agent is capture compound 1 Is allowed to join. Conversely, E) Interfering agent 1 that has an affinity for capture compound 1 (but no affinity for capture compound 2) does not bind to capture compound 2, so that the specific agent binds to capture compound 2. And F) Interfering agent 2 binds to capture compound 2 and thus prevents specific agents from binding. When G) and H) capture compounds 1 and 2 are used in a 1: 1 ratio, for example, the false signal reduction induced by interfering agent 1 or interfering agent 2 will be reduced to approximately 50%. The test principle of the competition test regarding an anti-hepatitis core (HBc) antigen antibody is shown. HBc: Hepatitis B core antigen; α-HBc: anti-HBc antigen antibody; unconjugated α-HBc antibody shown in double perimeter is an antibody that is potentially present in patient-derived samples and has a single perimeter The conjugated antibody shown in is the α-HBc antibody added during the assay; the conjugation is either Ru: ruthenium complex or bio: biotin: strp: streptavidin on solid support It is a coating. A) In the absence of analyte (α-HBc antibody), both Ru-conjugated and bio-conjugated α-HBc antibodies can bind to HBc added to the assay mixture. Through bio-strp interaction, the complex binds to the solid support and, after washing, the signal resulting from Ru conjugation can be measured. B) In the presence of the analyte (α-HBc antibody), αHBc-bio antibody and Ru conjugated antibody are prevented from binding to HBc added to the assay mixture and are therefore complexed to the solid support. Body binding is hindered. Thus, after washing, the Ru complex will be washed away, so no signal from the Ru complex can occur. In FIG. 2, HBc as well as α-HBc-bio conjugate are part of the capture compound complex. The test principle of the competition test regarding an anti-hepatitis core (HBc) antigen antibody is shown. HBc: Hepatitis B core antigen; α-HBc: anti-HBc antigen antibody; unconjugated α-HBc antibody shown in double perimeter is an antibody that is potentially present in patient-derived samples and has a single perimeter The conjugated antibody shown in is the α-HBc antibody added during the assay; the conjugation is either Ru: ruthenium complex or bio: biotin: strp: streptavidin on solid support It is a coating. A) In the absence of analyte (α-HBc antibody), both Ru-conjugated and bio-conjugated α-HBc antibodies can bind to HBc added to the assay mixture. Through bio-strp interaction, the complex binds to the solid support and, after washing, the signal resulting from Ru conjugation can be measured. B) In the presence of the analyte (α-HBc antibody), αHBc-bio antibody and Ru conjugated antibody are prevented from binding to HBc added to the assay mixture and are therefore complexed to the solid support. Body binding is hindered. Thus, after washing, the Ru complex will be washed away, so no signal from the Ru complex can occur. In FIG. 2, HBc as well as α-HBc-bio conjugate are part of the capture compound complex. 1 schematically illustrates the principles of the present invention as applied to a double antigen sandwich (DAGS) format. a: Analyte, d: detection compound, c1: capture compound 1, c2: capture compound 2, i1: interference agent 1, i2: interference agent 2. A) In the absence of analyte, the analyte will not bind to capture compound 1 (or 2) immobilized on the solid support, and therefore the detection compound will be washed away in the washing step. B) In the presence of an analyte, eg an antibody, the analyte binds to capture compound 1 and to the detection compound, thus mediating the binding of the detection compound to the solid surface. C) In the presence of interfering agent 1, which binds to capture compound 1 and detection compound but not to capture compound 2, the detection compound is immobilized on the solid support through interaction with interfering agent 1. Become. D) In the presence of interfering agent 2, which binds to capture compound 2 and detection compound but not to capture compound 1, the detection compound is not immobilized on the solid support through interfering agent 1. Conversely, E) in the presence of interfering agent 1, the detection compound is not immobilized on the solid support through interaction with interfering agent 2; and F) in the presence of interfering agent 2, the detection compound is captured. It will be immobilized on a solid support through compound 2. When G) and H) capture compounds 1 and 2 are used, for example in a 1: 1 ratio, the increase in spurious signal induced by interfering agent 1 or interfering agent 2 will be reduced to approximately 50%. The scheme can also be modified with respect to the use of two non-identical detection compounds, with changes to be made. 1 schematically illustrates the principles of the present invention as applied to a double antigen sandwich (DAGS) format. a: Analyte, d: detection compound, c1: capture compound 1, c2: capture compound 2, i1: interference agent 1, i2: interference agent 2. A) In the absence of analyte, the analyte will not bind to capture compound 1 (or 2) immobilized on the solid support, and therefore the detection compound will be washed away in the washing step. B) In the presence of an analyte, eg an antibody, the analyte binds to capture compound 1 and to the detection compound, thus mediating the binding of the detection compound to the solid surface. C) In the presence of interfering agent 1, which binds to capture compound 1 and detection compound but not to capture compound 2, the detection compound is immobilized on the solid support through interaction with interfering agent 1. Become. D) In the presence of interfering agent 2, which binds to capture compound 2 and detection compound but not to capture compound 1, the detection compound is not immobilized on the solid support through interfering agent 1. Conversely, E) in the presence of interfering agent 1, the detection compound is not immobilized on the solid support through interaction with interfering agent 2; and F) in the presence of interfering agent 2, the detection compound is captured. It will be immobilized on a solid support through compound 2. When G) and H) capture compounds 1 and 2 are used, for example in a 1: 1 ratio, the increase in spurious signal induced by interfering agent 1 or interfering agent 2 will be reduced to approximately 50%. The scheme can also be modified with respect to the use of two non-identical detection compounds, with changes to be made.

Example 1
Cloning and purification of recombinant hepatitis B core antigen A synthetic gene encoding hepatitis B core antigen (HBcAg) was purchased from Eurofins MWG Operan (Ebersberg, Germany). The following cloning steps were performed based on the pET24a expression plasmid from Novagen (Madison, Wis., USA). The vector was digested with BamH1 and Xho1 and a cassette containing HBcAg was inserted. The resulting plasmid insert was sequenced and found to encode the desired protein. The amino acid sequence of the resulting protein is shown in the sequence protocol of the present invention. Recombinant HBcAg did not contain a C-terminal hexahistidine tag.

  Recombinant HBcAg was purified according to the following protocol. E. coli BL21 (DE3) cells harboring the expression plasmid are grown in LB medium supplemented with kanamycin (30 μg / ml) until an OD600 of 1, and at a growth temperature of 37 ° C., isopropyl- Cytoplasmic overexpression was induced by adding β-D-thiogalactoside (IPTG) to a final concentration of 1 mM. After 4 hours incubation, cells were harvested by centrifugation (5000 xg for 20 minutes), frozen and stored at -20 ° C. For cell lysis, frozen pellets were prepared from 25 mM sodium phosphate pH 8.5, 6 mM MgCl 2, 10 U / ml Benzonase®, 1 tablet Complete® and 50 tablets of Complete® Resuspended in EDTA free (protease inhibitor cocktail) and the resulting suspension was lysed by high pressure homogenization. The crude lysate was centrifuged and the HBcAg in the supernatant was precipitated with ammonium sulfate (35% w / v). After further centrifugation, the precipitate was resuspended and dialyzed against phosphate buffer, followed by a heating step (30 minutes at 70 ° C.). After centrifugation, the clear supernatant was applied onto a Toyopearl DEAE 650-11 column (Tosoh Bioscience) pre-equilibrated with 25 mM potassium phosphate pH 7. The protein was then eluted by applying a gradient to a concentration of 500 mM potassium chloride. Finally, the protein was subjected to size exclusion chromatography (S400) and the protein containing fractions were pooled.

Example 2
Immunoassay to detect anti-HBc antibodies in a competitive test format Serum samples from a healthy blood donor panel were analyzed in an automated Cobase® analyzer (Roche Diagnostics GmbH) with respect to the presence of anti-HBc antibodies. Analyzed in HBc ECLIA (electrochemiluminescence immunoassay). Cobas e (registered trademark) and Elecsys (registered trademark) are registered trademarks of the Roche group. Samples were tested negative for anti-HBc in at least one CE-marked anti-HBc assay, which is different from the Elecsys® anti-HBc assay and is commercially available.

  In the assay, serum is reacted with HBc. Both a biotinylated antibody that is specific for HBcAg and a ruthenium complex (Tris (2,2′-bipyridyl) ruthenium (II) complex; (Ru (bpy) 32+) labeled antibody were added along with streptavidin-coated microparticles. Later, binding sites that were still free on the HBc antigen are occupied.The entire complex binds to the solid phase through the interaction of biotin and streptavidin.After removing unbound material, the voltage is applied to the electrode. After application and excitation with a platinum electrode, chemiluminescence at 620 nm is induced, which is measured by a photomultiplier tube.

  A high measurement indicates binding of the added biotinylated antibody and ruthenium complex labeled antibody, and thus indicates the absence of anti-HBc antibody in the sample. In the presence of anti-HBc antibodies in the sample, these antibodies compete with both types of assay-specific antibodies for binding to the antigen HBcAG, leading to a decrease in light emission at 620 nm after excitation with a platinum electrode. . The signal output is an arbitrary light unit.

  Thus, a sample having a measured value higher than the cut-off index 1.0 is considered non-reactive, while a sample having a measured value lower than or equal to the cut-off index 1.0 is considered reactive.

Three different HBcAg settings were tested based on a competitive Elecsys® anti-HBc assay format. “HBcAg X” refers to the antigen comprising SEQ ID NO: 2; “HBcAg Y” refers to the antigen comprising SEQ ID NO: 1. Only the antigen setting was modified and all other reagents and conditions were unchanged. True positive (infected) sample results were not affected by applying a combination of HBcAg X and Y as the first and second capture compounds (data not shown). Table 1 relates only to inconsistent (= false) positive results for the sample panel. The three different settings were as follows:
A) HBcAg X is used as a target in a competitive anti-HBc assay.
B) HBcAg Y (slightly different from HBcAg X) is used as a target in a competitive anti-HBc assay.
C) A combination of HBcAg X and HBcAg Y is used as a target in a competitive anti-HBc assay.

  Table 1 shows the results of competitive anti-HBc-ECLIA (electrochemiluminescence immunoassay). A commercially available serum (Bavarian Red Cross) that is negative for antibodies to the HBc antigen was used as the sample. Table 1 lists only the results with inconsistent findings. Different HBcAG showed individual patterns of inconsistent positive anti-HBc results. When using two different preparations of HBc antigen (HBcAG), 11 samples were (false) positive (reactive) results when using HBcAG X, only when using the second HBcAG Y , 7 samples were (false) positive results, and 2 samples were (false) positive results when both HBcAG X and HBcAG Y were used. In contrast, when using both antigens, a 1: 1 mixture of HBcAG X and HBcAG Y, only 5 of these 18 samples gave positive results and the number of false positive tests was reduced by more than 3 fold. . Specifically, when using the aforementioned 1: 1 mixture of both antigens (HBcAG X and HBcAG Y), the number of samples that were false positive results using HBcAGX alone was reduced from 11 to 3, and HBcAGY The number of samples that were false positive results using only 9 decreased from 9 to 5. As expected, the two samples that had false positive results with both antigens (sample numbers 2942 and 3657) also had false positive results with the mixture.

In conclusion, the specificity of this competitive immunoassay was increased when at least two slightly different HBc antigens were applied as a mixture.
By applying the principle of using two slightly different first and second capture compounds for the analyte in a method based on a competitive test format, the specificity is significantly increased, i.e. false positive results to a considerable extent It can be avoided.

TABLE 1 Competitive anti-HBc ECLIA (electrochemiluminescence immunoassay) results COI: Cut-off index

Claims (15)

A method for determining an analyte in a sample suspected of containing an analyte comprising: a) contacting the sample with at least first and second capture compounds for the analyte, wherein the first and second The second capture compound is a non-identical capture compound that is measurably different in at least one chemical and / or physical property that is not a property related to binding of the capture compound to a solid surface, the capture compound being the analysis Competing in binding to the object, and the capture compound is a polypeptide;
b) contacting the capture compound contacted with the sample with a specific agent, wherein the specific agent competes with the analyte for binding to the capture compound;
c) determining the amount of complex comprising the specific agent and capture compound; and d) determining the analyte in the sample based on the result of step c).   2. The method of claim 1, wherein the competition in binding to the molecule is binding to essentially the same or the same substructure of the molecule.   3. The method of claim 1 or 2, wherein said non-identical capture compound is a polypeptide, preferably an HB core (HBc) antigen. Said second capture compound is at least one of the following:
(I) introducing at least one amino acid exchange into the amino acid sequence of said first capture compound;
(Ii) producing the second capture compound in a different cell background when compared to the first capture compound;
(Iii) removing or preventing glycosylation of the second capture compound as compared to the first capture compound;
(Iv) purifying said second capture compound by different means and / or by different methods when compared to said first capture compound;
(V) denature and / or refold the second capture compound under different conditions when compared to the first capture compound; and (vi) the first capture compound. When derived from or can be derived from said first capture compound by storing said second capture compound under different conditions when compared to the method of.   The method according to any one of claims 1 to 4, wherein the analyte is an antibody against a viral antigen.   6. The method according to any one of claims 1 to 5, wherein step a) comprises contacting a few capture compounds for the analyte with the sample.   7. The method of any one of claims 1-6, wherein the first and second capture compounds do not compete for binding to interfering compounds.   A method for improving the specificity of an indirect immunoassay for determining an analyte comprising replacing 10% to 90% of a capture compound with a non-identical capture compound Wherein said method competes with the introduced capture compound for binding to said analyte.   9. The method of any one of claims 1-8, wherein the chemical structure in the capture compound that binds to the analyte is the same in all of the non-identical capture compounds. (I) at least two non-identical capture compounds that are measurably different in at least one chemical and / or physical property that is not a property related to binding of the capture compound to a solid surface, wherein the capture compound Competes for binding to the analyte and the capture compound is a polypeptide; or (ii) at least two non-identical detection compounds for the analyte that compete for binding to the analyte A kit for detecting an analyte in a sample containing a compound.   11. The kit of claim 10, further comprising a solid support for immobilizing the capture compound, or at least a component of the sample comprising the analyte. (I) at least two non-identical capture compounds that are measurably different in at least one chemical and / or physical property that is not a property related to binding of the capture compound to a solid surface, wherein the capture compound Competes for binding to the analyte and the capture compound is a polypeptide; or (ii) at least two non-identical detection compounds for the analyte, wherein the detection compound competes for binding to the analyte And a device for determining an analyte in a sample, including any means for determining the optical or / and enzymatic properties of an indicator contained in the detection compound. (I) at least two non-identical capture compounds that are measurably different in at least one chemical and / or physical property that is not a property related to the binding of the capture compound to a solid surface, for use in diagnosis of a disease, Wherein the capture compound competes for binding to the analyte and the capture compound is a polypeptide; or (ii) at least two non-identical detection compounds for the analyte indicative of disease, wherein the detection The compound competes for binding to the analyte,
A composition comprising:   14. A composition for use according to claim 13, wherein the disease is viral hepatitis.   Use of a composition comprising first and second capture compounds for determining an analyte in a sample, wherein the first and second capture compounds are properties relating to binding of the capture compound to a solid surface. A non-identical compound that is measurablely different in at least one chemical and / or physical property, wherein the capture compound competes for binding to the analyte and the capture compound is a polypeptide There is said use.